Currently, the best means of preventing colorectal cancer is through early detection of pre-neoplastic lesions in the colon through various non-invasive or invasive screening techniques. For example, all individuals age 50 and above in the United States are recommended to undergo some form of colorectal cancer screening on a regular basis. Those with personal or family histories that suggest a greater risk for the development of this problem should be screened at earlier ages and more intensively. However, the earliest recognizable abnormality that clearly indicates an increased risk for colorectal cancer development is the adenoma. Not only are individuals with adenomas at increased risk but with appropriate surveillance and preventive maneuvers, these persons have the greatest chances of benefiting from preventive strategies to inhibit the development of a malignancy. Thus, these individuals are ideal candidates for dietary or pharmacological interventions aimed at further reducing their risk.
Screening techniques have their limitations: 1) they require repetitive application even in individuals who are at only marginally increased risk for colon cancer (i.e., because of advanced age alone); and 2) the time interval between development of recurrent polyps (or a first polyp after a normal index endoscopic examination) or other pre-neoplastic lesions is long, making these macroscopic structural changes slow and inefficient biomarkers for risk assessment. For example, an individual at 50 years of age with a normal colonoscopy still may be at risk for colon cancer development and requires continued periodic screening for this problem. Furthermore, this individual may or may not benefit from chemopreventive intervention. A simple solution is to accept that all individuals are candidates for screening programs and chemopreventive interventions. However, in practice, this is: a) technically impossible; b) associated with potentially excessive risk compared to the benefits obtained; and c) is likely too expensive to be worthwhile. Thus, the ability to predict definitively whether an individual is at increased risk for the development of colon cancer would be a great advance that would target those in need of intervention and, therefore, save lives and reduce health care expenses.
Apoptosis is a process whereby cells die in a characteristic, controlled manner in response to specific stimuli and according to an intrinsic genetic program. Interference in the process of apoptosis, therefore, is believed to be important for the development of neoplasia. For example, the mechanism of induction of neoplasia by the protooncogene bcl-2 is believed to reside specifically in the inhibition of apoptosis (see Baretton et al., 1996, Cancer 77:255-264). Certain carcinogens may induce neoplasia by interfering directly with apoptosis (see, for example, Hayashi et al., 1996, Cancer Research 56:4307-4310).
In the mammalian gastrointestinal tract, apoptosis appears to be the process principally regulating cell loss (Hall et al., 1994, J. Cell Science 107:3569-3577). Recent reports, for example, Tsujitani et al. (1996, Cancer 77:1711-1716) claim that increased apoptosis is characteristic of colorectal neoplasia. Morin et al. (1996, Proc. Nat. Acad. Sci. U.S.A. 93:7950-7954) suggested that apoptosis plays a role in both advanced colorectal tumors but also in the earliest stages of neoplasia. Arai and Kino (1995, J. Pathol. 176:37-44) report that reduced prerentage of apoptotic cells in human colorectal tissue may lead to neoplasia. These conflicting reports on the level of apoptosis in relation to neoplasia may relate to the stage of neoplasia and the location from which the biopsy is taken.
Methods for the measurement of apoptosis in a cellular or tissue sample have been described. Numerous methods have been proposed in the literature and commercial kits exists for measurement of apoptosis. A quantitative measure of apoptosis in a cellular or tissue sample can be obtained by determining the ratio of the number of apoptotic cells in the sample to the total number of cells in the sample, the ratio being referred to as the apoptotic index. Morphologically, apoptotic cells display nuclear chromatin condensation, compactness of cytoplasmic organelles, and the appearance of pedunculated protuberances on the cell surface. DNA fragmentation characteristic of apoptotic cells can be identified by isolating nuclear DNA and analyzing it using gel electrophoresis to identify strand breakage. Using flow cytometry on cells stained with a specific DNA stain such as propidium iodide, the percent of apoptotic cells can be assessed by counting the number of cells in a sample with a DNA content typical of apoptotic cells. Histologically, apoptotic cells can be identified after staining by the terminal deoxynucleotidyl transferase dUTP-biotin nick end labeling (TUNEL) method, or a modification thereof, which labels fragmented DNA. Biotin-dUTP added to DNA is detected with avidin-peroxidase or other well-known methods.
Methods for assessing risk of development of colorectal neoplasia based upon assessment of apoptosis from a biopsy specimen have been described, but such methods have relied on in vitro cultivation of such cells with or without an additional treatment of the cells with agents in order to assess apoptosis. For example, Bedi et al. (1995, Cancer Research 55:1811-1816) cultivate colorectal epithelial cells in vitro prior to performing an assay for apoptosis. Garewal et al. (1996, Cancer Research 56:1480-1483) measure the ability for bile acids to induce apoptosis in vitro in colorectal mucosal cells. Payne et al. (1995, Ultrastruct. Pathol. 19:221-248) suggested that a resistance-to-apoptosis bioassay may prove useful as a biomarker for individuals at risk for colon cancer, as they proposed that the presence of bile salts that accompany a high-fat diet may select for apoptosis-resistant epithelial cells in the colon. Such in vitro methods and bioassays are cumbersome. Notwithstanding the skepticism proposed by Einspahr et al. (1997, Cancer Epidemiology, Biomarkers & Prevention 6:37-48) who surmised that quantitation of apoptotic cells within colonic crypts will be difficult, there exists a need for a simple, rapid method for assessing apoptosis directly from colorectal biopsy specimens to determine whether an individual has or has had colorectal neoplasia or has a risk for the development of colorectal neoplasia. Furthermore, there exists a need to identify individuals with or at risk for colorectal neoplasia who are or will be-responsive to prevention or intervention, whether by drug therapy or dietary modification, by monitoring the change in the extent of apoptosis in a biopsy specimen obtained at an interval or intervals after the individual has initiated prevention or intervention therapy.